The present invention relates to a substantially leak-proof closure system for storing fluids under cold storage conditions, where the closure system includes a container component and a cap component which can be fitted onto the container component.
All references referred to herein are hereby incorporated by reference in their entirety. The incorporation of these references, standing alone, should not be construed as an assertion or admission by the inventors that any portion of the contents of all of these references, or any particular reference, is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the inventors reserve the right to rely upon any of such references, where appropriate, for providing material deemed essential to the claimed invention by an examining authority or court. No reference referred to herein is admitted to be prior art to the claimed invention.
Procedures for determining the presence or absence of specific organisms or viruses in a test sample commonly rely upon nucleic acid-based probe testing. To increase the sensitivity of these tests, an amplification step is often included to increase the number of potential nucleic acid target sequences present in the test sample. During amplification, polynucleotide chains containing the target sequence or its complement are synthesized in a template-dependent manner from ribonucleoside or deoxynucleoside triphosphates using nucleotidyltransferases known as polymerases. There are many amplification procedures in common use today, including the polymerase chain reaction (PCR), Q-beta replicase, self-sustained sequence replication (3SR), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA) and loop-mediated isothermal amplification (LAMP), each of which is well known in the art. See, e.g., Mullis, xe2x80x9cProcess for Amplifying Nucleic Acid Sequences,xe2x80x9d U.S. Pat. No. 4,683,202; Erlich et al., xe2x80x9cKits for Amplifying and Detecting Nucleic Acid Sequences,xe2x80x9d U.S. Pat. No. 6,197,563; Walker et al., Nucleic Acids Res., 20:1691-1696 (1992); Fahy et al., xe2x80x9cSelf-sustained Sequence Replication (3WSR): An Isothermal Transcription-Based Amplification System Alternative to PCR,xe2x80x9d PCR Methods and Applications, 1:25-33 (1991); Kacian et al., xe2x80x9cNucleic Acid Sequence Amplification Methods,xe2x80x9d U.S. Pat. No. 5,399,491; Davey et al., xe2x80x9cNucleic Acid Amplification Process,xe2x80x9d U.S. Pat. No. 5,554,517; Birkenmeyer et al., xe2x80x9cAmplification of Target Nucleic Acids Using Gap Filling Ligase Chain Reaction,xe2x80x9d U.S. Pat. No. 5,427,930; Marshall et al., xe2x80x9cAmplification of RNA Sequences Using the Ligase Chain Reaction,xe2x80x9d U.S. Pat. No. 5,686,272; Walker, xe2x80x9cStrand Displacement Amplification,xe2x80x9d U.S. Pat. No. 5,712,124; Notomi et al., xe2x80x9cProcess for Synthesizing Nucleic Acid,xe2x80x9d U.S. Pat. No. 6,410,278; Dattagupta et al., xe2x80x9cIsothermal Strand Displacement Amplification,xe2x80x9d U.S. Pat. No. 6,214,587; and HELEN H. LEE ET AL., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES: APPLICATION TO DISEASE DIAGNOSIS (1997).
Because polymerase activity is readily lost at ambient temperature, it is common to manufacture amplification kits which include polymerase-containing enzyme reagents that have been freeze-dried in formulations containing other necessary co-factors and substrates for amplification. See, e.g., Shen et al., xe2x80x9cStabilized Enzyme Compositions for Nucleic Acid Amplification,xe2x80x9d U.S. Pat. No. 5,834,254. It is also common to manufacture amplification kits which include amplification reagents containing nucleoside triphosphates and/or amplification primers in freeze-dried formulations. Alternatively, these enzyme and amplification reagents can be kept in cold storage at temperatures well below 0xc2x0 C. (e.g., at about xe2x88x9220xc2x0 C.). An advantage of cold storage is that reagents can be manufactured and shipped directly on dry ice to the end user, avoiding lengthy and expensive lyophilization procedures prior to shipping, as well as time-consuming and exact reconstitution procedures by the end-user. However, storing fluid reagents in laboratory freezers is generally disfavored because these reagents, which may contain, for example, glycerol or non-ionic detergents (non-ionic detergents can be used to sequester ionic detergents in a sample solution which may solubilize target nucleic acid or interfere with enzyme function and are often used to stabilize the enzymes), tend to remain highly viscous fluids in commonly used sub-zero freezers.
As the volume of these highly viscous fluids expands under cold storage conditions, one leak theory provides that a significant meniscus forms and rises which, if high enough, can seep through the seals of conventional storage containers. Other leak theories relate to temperature fluctuations due to the repeated opening and closing of storage freezers. According to one of these theories, it is believed that the stored fluid freezes and water is removed from the frozen fluid by sublimation which settles, inter alia, in the interstices between the cap and the container. When the storage freezer is subsequently opened, the temperature within the freezer rises and the water vapor forms a condensate which freezes as the storage freezer is restored to its normal operating temperature. As the condensate freezes, it expands in the interstices between the cap and the container, thereby weakening the seal. Another of these theories provides that the stored fluid does not freeze, but the opening and closing of the storage freezer causes temperature fluctuations which lead to the formation of a condensate in the interstices between the cap and the container. Like the sublimation theory, the freezing of this condensate as the storage freezer is restored to its normal operating temperature could result in sufficient expansion between the cap and the container to create fissures which might provide an avenue of escape for fluid stored in the container.
Besides wasting expensive reagents, seepage of reagents from their storage containers is especially problematic when the reagents have been aliquoted for use in a specified number of amplification reactions in an automated instrument. (See Ammann et al., xe2x80x9cAutomated Process for Isolating and Amplifying a Target Nucleic Acid Sequence,xe2x80x9d U.S. Pat. No. 6,335,166, for an example of an instrument for performing automated nucleic acid amplification and detection steps.) Therefore, loss of some reagent from the container could affect amplification efficiency in one or more assays.
Consequently, it would desirable to have a closure system that provides a sealing system which prevents or severely limits seepage of a stored fluid substance under cold storage conditions, especially substances which remain at least partially fluid under those cold storage conditions. Such substances may include one or more components affecting the viscosity or surface tension of the stored fluid or which contribute to freezing point depression of the stored fluid. In particular, the desired closure system would be useful for storing enzyme and/or amplification reagents for use in a nucleic acid amplification reaction, where the reagents are stored in a conventional laboratory freezer at a temperature of about xe2x88x9220xc2x0 C. To accommodate its use in an automated instrument, the closure system should preferably be designed so that its internal volume is maximized and so that a robotic pipettor will have access to all or nearly all of the full volume of the stored fluid reagent.
The present invention meets this need by providing a substantially leak-proof closure system for storing fluids under cold temperature conditions which includes a container and a cap. The container component, which is generally cylindrical in shape, includes a side wall having inner and outer surfaces, a closed bottom end and an open top end having an annular top rim and a beveled lip which depends inward from the inner circumference of the top rim. The cap component includes a top wall having a generally circular shape, an annular outer skirt which depends from the periphery of the top wall and has an inner surface adapted to grip the outer surface of the top end of the container (e.g., mated helical threads or snap-fit arrangement), and an annular inner skirt which depends from the top wall and has an outer surface which comprises a lower seal bead and an upper beveled portion which is mated with the beveled lip of the container. The seal bead of the inner skirt is sized and arranged to be in sealing contact with the inner surface of the top end of the container when the cap is fitted onto the container. By xe2x80x9csealing contactxe2x80x9d is meant an interference force fit between the seal bead of the cap and the inner surface of the container. Additionally, the upper beveled portion of the cap and the beveled lip of the container are engaged in an interference fit when the cap is fitted onto the container. The interference fit of this closure system is expected to provide a substantially leak-proof sealing system under cold storage conditions. As used herein, xe2x80x9ccold storage conditionsxe2x80x9d refers to conditions under which water freezes.
In one embodiment of the present invention, the outer surface of the inner skirt is configured so that an annular air pocket is formed between the outer surface of the inner skirt and the inner surface of the container and between the seal bead and the upper beveled portion when the cap is fitted onto the container. This configuration permits greater deflection of the seal bead as the inner skirt is inserted into the container, thereby increasing the load of the seal bead on the inner surface of the container and, thus, reducing the opportunity for fluid leakage. Preferably, the outer surface of the inner skirt has a generally arcuate shape between the beveled portion and the seal bead, and an inner surface of the inner skirt has a generally cylindrical shape.
In another embodiment of the present invention, a bottom surface of the inner skirt is rounded or beveled so that a rising meniscus in the closure system may be at least partially diverted into an area defined by the inner surface of the inner skirt under cold storage conditions. In this way, the forces exerted by an expanding fluid may be substantially equilibrated on both sides of the bottom surface of the inner skirt or, preferably, those forces exerted by the expanding fluid on the inner surface of the inner skirt will exceed those forces exerted on the outer surface of the inner skirt.
In yet another embodiment of the present invention, the inner surface of the container adjacent to and below the beveled lip includes a substantially no draft region (i.e., a region which is not tapered relative to the longitudinal axis of the container), and the seal bead sealingly contacts the inner surface in the no draft region when the cap is fitted onto the container. To further improve the seal between the seal bead and the no draft region, the core pin used to form the container during an injection molding procedure is preferably given a radial polish and, in the no draft region, hand-lapped prior to injection molding to prevent the formation of draw and sink lines on the inner surface of the molded container, especially in the no draft region. In this embodiment, the outer diameter of the seal bead is preferably smaller than the inner diameter of the top rim and greater than the inner diameter of the no draft region to facilitate the formation of a seal between inner surface of the top end of the container and the outer surface of the inner skirt of the cap.
In still another embodiment of the present invention, the closure system is provided with a solution having added thereto at least one component which contributes to freezing point depression of the solution (e.g., a salt), increases the viscosity of the solution (e.g., ethylene glycol, glycerol or dextran), or alters the surface tension of the solution (e.g., a detergent, surfactant or oil). Such solutions may further include one or more enzyme reagents (e.g., RNA or DNA polymerase) for use in amplifying a nucleic acid sequence of interest. Enzyme reagents for use in performing a transcription-based amplification, for example, include reverse transcriptase and RNA polymerase. Other amplification reagents may also be included, such as, for example, amplification oligonucleotides (e.g., primers, promoter-primers and/or splice templates), nucleotide triphosphates, metal ions and co-factors necessary for enzymatic activity. The reagents are preferably provided in buffered formulations such as, for example, formulations comprising 0.01% (v/v) TRITON(copyright) X-100, 41.6 mM MgCl2, 1 mM ZnC2H3O2, 10% (v/v) glycerol, 0.3% (v/v) ethanol, 0.02% (w/v) methyl paraben, and 0.01% (w/v) propyl paraben. Other solutions which can be formulated for use in an amplification procedure will be readily appreciated by those skilled in the art.
These and other features, aspects, and advantages of the present invention will become apparent to those skilled in the art after considering the following detailed description, appended claims and accompanying drawings.